The mechanical interactions between cells and extracellular matrix (ECM) drive fundamental processes such as developmental morphogenesis, wound healing, and the organization of bioengineered tissues. This project is focused on investigating how these interactions regulate corneal keratocyte mechanical behavior and its role in corneal transparency, through the development of novel 3-D culture models, and the application of quantitative 3-D and 4-D imaging techniques. Research conducted in the prior grant period has provided important insights into the regulation of corneal keratocyte spreading and migration within 3-D matrices. We demonstrated that corneal keratocytes differentiate into distinct mechanical phenotypes (dendritic vs. contractile) in response to specific growth factors expressed during wound healing, and that this process is regulated, in part, by the balance between Rho and Rac activation and the mechanical stiffness of the ECM. We also demonstrated for the first time that whereas corneal fibroblasts generally move independently within collagen matrices, fibrin induces a switch to an interconnected, collective mode of cell spreading and migration, which is associated with localized fibronectin secretion, cadherin expression and development of intracellular stress fibers. Using an in vivo HRT- RCM confocal microscope that we custom-modified to allow quantitative full-thickness corneal imaging, we also generated pilot data suggesting for the first time that dendritic migration can occur in vivo following mechanical scrape injury in the rabbit, in which healing occurs without significant loss of transparency. In contrast, a collective, fibroblastic morphology was observed following transcorneal freeze injury (which leads to increased cellular light scattering and fibrosis). Corneal myofibroblasts have also been shown to organize into an interconnected mesh following incisional surgery or photorefractive keratectomy (PRK). In the current application, we will further characterize the mechanisms regulating corneal keratocyte mechanical behavior in vitro (in 3-D culture), and correlate these with wound healing phenotypes observed in vivo. Specific Aim 1 will establish how growth factors modulate cell contractility, matrix reorganization production of normal and fibrotic ECM components and ALDH1A1 expression levels in collagen and fibrin matrices of varying stiffness, and determine whether these expression profiles are modulated by inhibiting Rho kinase. These studies will be the first to determine how matrix composition and stiffness, growth factors and Rho kinase activation regulate expression of normal and fibrotic keratocyte markers and corneal crystallins in 3-D culture; factors that are directly associated with maintenance or loss of corneal transparency during in vivo wound healing. Specific Aim 2 will determine the dependency of collective cell migration on growth factors, expression and localization of fibronectin, 51 integrin and cadherin, ECM degradation and patterning, and Rho kinase-mediated cell contractility, using both quiescent corneal keratocytes and activated dermal and corneal fibroblasts. Corneal fibroblasts and myofibroblasts have previously been shown to form an interconnected network during healing after incisional surgery, lamellar keratectomy and PRK. Our unique model for studying collective cell migration in 3-D culture should provide important new insights into the mechanical and biochemical regulation of this fundamental process. Specific Aim 3 will in vivo confocal imaging and immunocytochemistry to correlate cell morphology, connectivity and backscattering with expression of fibroblast and myofibroblast markers after mechanical scrape, transcorneal freeze and lamellar keratectomy in the rabbit, and determine whether these responses can be modulated by inhibiting Rho kinase (using Y-27632). Fibroblast and myofibroblast transformation of quiescent corneal keratocytes lead to corneal haze development following injury or surgery. Our research suggests that Y-27632 is a natural candidate for inhibiting this transformation in vivo (possible bench to beside translation of a new therapy). During the course of the grant, training will be provided to a total of two biomedical engineering graduate (PhD) students, two post-docs, as well as 4 summer medical students and 3 undergraduate research fellows.